Volume 18, Issue 2, Pages 253-261 (April 2005) Single Protein Production in Living Cells Facilitated by an mRNA Interferase  Motoo Suzuki, Junjie Zhang, Mohan Liu, Nancy A. Woychik, Masayori Inouye  Molecular Cell  Volume 18, Issue 2, Pages 253-261 (April 2005) DOI: 10.1016/j.molcel.2005.03.011 Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 1 Expression of Human Eotaxin in E. coli with and without MazF Coexpression (A) The amino acid sequence of the human eotaxin fusion. The sequence below it represents that of the mRNA derived from the synthetic eotaxin gene (designed with preferred E. coli codons). Mature, proteolytically processed eotaxin comprises 74 residues from G18 (shown here right after the NdeI site) from P91. Triplets highlighted in black were changed to ACA for the experiment shown in the left panel of Figure 2C. (B) The effect of MazF expression on eotaxin production. Upon reaching an OD600 of 0.5, cultures of E. coli BL21(DE3) transformed with pACYCmazF and pColdI(SP-1)eotaxin (right), pACYCmazF alone (left), or pColdI(SP-1)eotaxin alone (middle) were shifted from 37°C to 15°C for 45 min. New protein synthesis was then monitored by isotopic labeling with [35S]-methionine for 15 min before (C control lane) or at intervals after (0–72 hr) IPTG induction. Equivalent amounts of cell lysate, derived from equal culture volumes, were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by autoradiography. Molecular weight markers on the left; the position of eotaxin designated by an arrow to the right. Molecular Cell 2005 18, 253-261DOI: (10.1016/j.molcel.2005.03.011) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 2 Effect of ACA Sequences on Expression of Human Eotaxin and Yeast Proteins (A) Cultures of E. coli BL21(DE3) transformed with pACYCmazF and either pColdI(SP-1)eotaxin (left) or pColdI(SP-2)eotaxin (right) were shifted from 37°C to 15°C for 45 min. New protein synthesis was then monitored by isotopic labeling with [35S]-methionine for 15 min before (C control lane) or at intervals after (0–96 hr) IPTG induction. Equivalent amounts of cell lysate, derived from equal culture volumes, were subjected to SDS-PAGE followed by autoradiography. Molecular weight markers for both panels are shown on the left. (B) Cells from an E. coli BL21(DE3) culture carrying pACYCmazF and pColdI(SP-2)eotaxin were harvested after cold shock and IPTG induction at the times indicated followed by SDS-PAGE and Coomassie blue staining. Equivalent amounts of cell lysate, derived from equal culture volumes, were loaded. The position of eotaxin in both panels is designated by the arrow to the right. (C)eotaxin genes without (right) and with five (left) ACA-encoding sequences were expressed in the pColdI(SP-1) vector in BL21(DE3) cells subjected to the same conditions described for Figure 1. The positions of the new ACA sequences are shown in Figure 1A. Molecular weight markers are on the left; the position of eotaxin designated by an arrow is to the right. (D and E) Wild-type (wt) and ACA-less yeast Hsp10 (D) and Rpb12 (E) mRNAs were expressed from pColdI(SP-2) along with wt MazF from pACYCmazF. Experiments were carried out as described for Figure 1. Molecular weight markers are on the left; the position of each target protein is designated by an arrow to the right. Molecular Cell 2005 18, 253-261DOI: (10.1016/j.molcel.2005.03.011) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 3 Expression of E. coli Proteins in the SPP System Wt and ACA-less EnvZB (A) and CspA (B) mRNAs were expressed from pColdI(SP-2) along with wt MazF from pACYCmazF. Expression was carried out as described for Figure 1, and experiments were followed by SDS-PAGE and Coomassie blue staining. Molecular weight markers are on the left; the position of each target protein is designated by an arrow to the right. Molecular Cell 2005 18, 253-261DOI: (10.1016/j.molcel.2005.03.011) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 4 Expression of the Inner Membrane Protein LspA with the SPP System (A) LspA expression in pColdIV(SP-2) relative to total cell protein synthesis was carried out as described in Figure 1 by using pACYCmazF(−9ACA). Molecular weight markers for all three panels are shown on the left. (B) LspA expression was profiled as in (A) but only membrane fractions were loaded for each time point. (C) Analysis of the membrane fraction components. The membrane fraction from (B) was further fractionated after LspA was induced for 1 hr to separate the inner from the outer membrane fractions. Lane 1, total cellular proteins; lane 2, the soluble fraction obtained after ultracentrifugation; lane 3, the membrane fraction obtained after ultracentrifugation; lane 4, sarkosyl soluble fraction (inner membrane fraction); and lane 5, sarkosyl insoluble fraction (outer membrane fraction). The position of LspA in all three panels is indicated by the arrow. Molecular Cell 2005 18, 253-261DOI: (10.1016/j.molcel.2005.03.011) Copyright © 2005 Elsevier Inc. Terms and Conditions